A broad-spectrum antimicrobial peptide AG3 and its preparation method and application

Abstract

The present invention provides a broad-spectrum antimicrobial peptide AG3 and a preparation method and application thereof, belonging to the field of biotechnology, and the amino acid sequence thereof is shown in SEQ ID No. 1. The antimicrobial peptide AG3 of the present invention has a significant inhibitory effect on 11 Gram-negative and Gram-positive bacteria and fungal species such as Escherichia coli, Pseudomonas aeruginosa, Salmonella pullorum, Staphylococcus aureus, and Candida albicans, and the average antibacterial activity reaches 2 μM; the bactericidal power also shows that it has a high bactericidal ability against bacteria; at the same time, its hemolytic activity is low, and the therapeutic index is as high as 32; in addition, it still has good antibacterial activity in physiological concentration salt ions, indicating that it has a high salt ion tolerance, and related mechanism experiments prove that it realizes antibacterial function through a physical membrane rupture mechanism.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a broad-spectrum antimicrobial peptide AG3 and a preparation method and application thereof. Background Art

[0002] In animal husbandry, in order to protect livestock and poultry from various diseases and improve their production performance, people generally add certain antibiotics to livestock and poultry feed. However, due to the wide coverage of animal husbandry and the coexistence of individual households and large factories, people cannot fully supervise the use of antibiotics, and the phenomenon caused by the abuse of antibiotics has received more and more attention. The emergence of microbial resistance caused by the abuse of antibiotics and the emergence of "super bacteria" are problems that cannot be ignored. The resistance of bacteria to antibiotics in the environment continues to grow, and the ecological environment is gradually becoming worse. It is urgent to find new, safe and reliable alternative products.

[0003] Antimicrobial peptides (AMPs) are an inherent component of the innate immune system. AMPs are widely present in almost all living organisms and provide the first line of defense against a wide range of invading microorganisms. However, compared with traditional antibiotics, most existing natural antimicrobial peptides have low bactericidal activity and single performance, which undoubtedly limits the application of antimicrobial peptides in actual production. Summary of the invention

[0004] Based on the above shortcomings, the purpose of the present invention is to provide an antimicrobial peptide AG3 with broad-spectrum antimicrobial activity, which solves the problems of the abuse of antibiotics leading to the generation of microbial resistance and the low bactericidal activity and single performance of natural antimicrobial peptides.

[0005] The technical solution adopted by the present invention is as follows: an antimicrobial peptide AG3 with broad-spectrum antimicrobial activity, whose amino acid sequence is shown in SEQ ID No.1, and whose C-terminus is -NH 2 Amidation.

[0006] Another object of the present invention is to provide a method for preparing the antimicrobial peptide AG3 with broad-spectrum antimicrobial activity as described above, comprising the following steps:

[0007] S1: Central symmetric α-helical peptide template: (y+hhh+y)n-NH 2 The design is based on , wherein h is a hydrophobic amino acid, + is a positively charged amino acid, the hydrophobic amino acids are Gly, Ala and Leu, and the positively charged amino acid is Lys, and the amino acid sequence of the obtained polypeptide is shown in SEQ ID No.1;

[0008] S2: using a solid phase chemical synthesis method to obtain a peptide resin using a peptide synthesizer, and then cutting the obtained peptide resin with TFA to obtain a polypeptide;

[0009] S3: After purification by reversed-phase high-performance liquid chromatography and identification by mass spectrometry, the antibacterial activity, antifungal activity, hemolytic activity, salt ion stability, bactericidal kinetics and antibacterial mechanism tests were performed to complete the preparation of the antimicrobial peptide, which was finally named antimicrobial peptide AG3.

[0010] Another object of the present invention is to provide a use of the antimicrobial peptide AG3 as described above in the preparation of a medicament for treating Gram-negative bacteria, Gram-positive bacteria or / and fungal infections.

[0011] Furthermore, the Gram-negative bacteria mentioned above are Escherichia coli, Salmonella typhimurium, Salmonella pullorum or Pseudomonas aeruginosa.

[0012] Furthermore, the Gram-positive bacteria mentioned above is Staphylococcus aureus.

[0013] Furthermore, the fungus mentioned above is Candida albicans.

[0014] Beneficial effects and advantages of the present invention: The antimicrobial peptide AG3 designed by the present invention has the advantages of broad antibacterial spectrum, high antibacterial activity and low toxicity. Antimicrobial peptide AG3 was tested for its antibacterial and hemolytic activity and found to have a significant inhibitory effect on 11 Gram-negative / positive bacteria and fungal species such as Escherichia coli, Pseudomonas aeruginosa, Salmonella pullorum, Staphylococcus aureus, and Candida albicans. The average antibacterial activity reached 2 μM, and the hemolytic activity was low. The therapeutic index was as high as 32, and it still had good antibacterial activity in physiological concentrations of salt ions, indicating that it has a high salt ion tolerance. In summary, antimicrobial peptide AG3 is an antimicrobial peptide with high application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the mass spectrum of antimicrobial peptide AG3;

[0016] Figure 2 is the chromatogram of antimicrobial peptide AG3;

[0017] Figure 3 is the hemolytic activity graph of AG3;

[0018] Figure 4 This is the bactericidal power diagram of the antimicrobial peptide AG3;

[0019] Figure 5 Outer membrane permeability of E. coli 25922 after treatment with antimicrobial peptide AG3;

[0020] Figure 6PI staining of E.coli 25922 after treatment with antimicrobial peptide AG3 DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0022] Example 1

[0023] Design of antimicrobial peptides: hydrophobic amino acids (Gly, Ala, Leu) and positively charged amino acids (Lys) in a central symmetric α-helical peptide template (y+hhh+y) n -NH 2 Based on the amino acid sequence of the antimicrobial peptide AG3, an antimicrobial peptide with broad-spectrum antimicrobial activity was designed.

[0024] Table 1 Amino acid sequence and main structural parameters of antimicrobial peptide AG3

[0025]

[0026]

[0027] Example 2

[0028] The solid phase chemical synthesis method synthesizes peptides. The specific steps are:

[0029] 1. The preparation of peptides is carried out one by one from the C-terminus to the N-terminus, and is completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of the peptide) is connected to Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); according to this procedure, it is synthesized from the C-terminus to the N-terminus in sequence until the synthesis is completed, and the peptide resin with the side chain protection of the Fmoc group removed is obtained.

[0030] 2. Add a cleavage reagent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter; wash with precipitated TFA (trifluoroacetic acid), mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, and then add about 10 times the volume of pre-cooled anhydrous ether, precipitate at -20°C for 3 hours, precipitate a white powder, centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain a polypeptide, wherein the cleavage reagent is a mixture of TFA, water and TIS (triisopropylchlorosilane) in a mass ratio of 95:2.5:2.5.

[0031] 3. Use 0.2 mol / L sodium sulfate (phosphoric acid adjusted to pH 7.5) to balance the column for 30 min, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, C18 reverse phase atmospheric pressure column, use gradient elution (eluent is methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30), flow rate is 1 mL / min, detection wave is 220 nm, collect the main peak, and freeze-dry; further purify using reverse phase C18 column, eluent A is 0.1% TFA / water solution; eluent B is 0.1% TFA / acetonitrile solution, elution concentration is 25% B to 40% B, elution time is 12 min, flow rate is 1 mL / min, collect the main peak as above, and freeze-dry.

[0032] 4. Identification of polypeptides: The antimicrobial peptides obtained above were analyzed by electrospray mass spectrometry. The molecular weight shown in the mass spectrum was basically consistent with the theoretical molecular weight in Table 1 (see Appendix Figure 1 ), the purity of the peptide is greater than 95% (see Appendix Figure 2 ).

[0033] Example 3

[0034] The prepared polypeptides were tested for in vitro antibacterial activity, hemolytic activity, and salt ion stability:

[0035] 1. Determination of antibacterial activity: Prepare the peptide into a certain storage solution for use. Determine the minimum inhibitory concentration of the antimicrobial peptide using the microbroth dilution method. Pick a single colony and inoculate it in MHB, culture it at 220rpm and 37℃ until the logarithmic growth phase, and adjust its concentration to OD with MHB under the condition of K=1.000. 600nm = 0.1, and finally diluted 1000 times with MHB to 0.5-1×10 5 CFU / mL; add 95μL 0.2% BSA diluent to row A of a 96-well plate, and add 50μL 0.2% BSA diluent to the remaining wells. Add 5μL of 2.56mM peptide to the wells in row A, mix thoroughly, then pipette 50μL to row B, and so on, dilute to row G in multiple proportions, pipette 50μL and discard after mixing. Set up three parallels for each peptide to be tested; add 50μL of bacterial solution to the wells in rows AG of a 96-well plate, add 50μL of bacterial solution to wells 1-6 in row H as a positive control, add 50μL of fresh MHB medium to wells 7-12 as a negative control, mix well and culture at 37°C for 24h. Measure the light absorbance at 492nm with an ELISA reader to determine the minimum inhibitory concentration.

[0036] 2. Determination of antifungal activity: The peptide was prepared into a certain storage solution for use. The minimum antifungal concentration of the antimicrobial peptide was determined by the microbroth dilution method. A single colony was picked in a potato liquid culture medium (containing chloramphenicol) and cultured at 170 rpm and 35°C until the logarithmic growth phase. The concentration was adjusted to OD 0.05 with PRMI-1640 culture medium under the condition of K = 1.000. 600nm =0.4, and finally diluted 1000 times with PRMI-1640 to 0.5-1×10 3 CFU / mL; the subsequent operation is the same as the antibacterial activity determination method; the difference is that fresh 1640 is used as a negative control, and after mixing, it is incubated at 35°C for 48 hours. The light absorption value is measured at 492nm using an ELISA reader to determine the minimum inhibitory concentration. The test results are shown in Table 2.

[0037] Table 2 Antimicrobial activity of antimicrobial peptide AG3

[0038]

[0039] It can be seen from Table 2 that the antimicrobial peptide AG3 has a high antibacterial activity against both Gram-negative and Gram-positive bacteria, and also has a certain antibacterial ability against Candida albicans.

[0040] 3. Determination of hemolytic activity: Collect 1 mL of fresh human blood, dissolve it in 2 mL of PBS solution after anticoagulation with heparin, and collect red blood cells after centrifugation at 1000g for 5 minutes; wash with PBS three times and resuspend with 10 mL PBS; take 50 μL of red blood cell suspension and 50 μL of antimicrobial peptide AG3 solution with different concentrations, mix them evenly, incubate in a 37°C incubator for 1 hour, take out, and centrifuge at 4°C and 1000g for 5 minutes; take the supernatant and measure the absorbance at 570nm with an enzyme marker; take the average value of each group and compare and analyze. 50 μL of red blood cells plus 50 μL PBS was used as a negative control; 50 μL of red blood cells plus 50 μL 0.1% Tritonx-100 was used as a positive control. The median lethal concentration (IC50) of blood cells was measured. 50 ) to determine the hemolytic activity of antimicrobial peptides. The test results are shown in the attached Figure 3 And Table 3.

[0041] Table 3 Therapeutic index of antimicrobial peptide AG3

[0042]

[0043] pass Figure 3 As can be seen from Table 3, the hemolytic activity of antimicrobial peptide AG3 is low within the detection range.

[0044] In order to analyze the clinical application potential of antimicrobial peptide AG3, the therapeutic index (IC 50As can be seen from Table 3, the antimicrobial peptide AG3 has a high therapeutic index, indicating that the designed AG3 has the potential to replace antibiotics in clinical practice.

[0045] 4. Salt ion stability determination: Add different concentrations of salt ions to the BSA peptide diluent, and determine the change in the MIC value of the antimicrobial peptide AG3 against E.coil25922 according to the above antimicrobial activity determination method. The final salt ion concentrations determined are: 150mM NaCl, 4.5mM KCl, 6μM NH 4 Cl, 8 μM ZnCl 2 , 1 mM MgCl 2 and 4 μM FeCl 3 The test results are shown in the table

[0046] Table 4 MIC values ​​(μM) of antimicrobial peptide AG3 against E.coil ATCC 25922 at physiological salt concentrations

[0047]

[0048] As can be seen from Table 4, the antimicrobial peptide AG3 still has good antimicrobial activity in physiological concentrations of salt ions, indicating that it has a high salt ion tolerance and has certain clinical application potential.

[0049] 5. Bactericidal kinetics determination: (1) Preparation of bacterial solution: Bacteria: Take the strain frozen at -20℃ and streak it on MHA solid medium and culture it at 37℃ overnight. Then pick a single colony and inoculate it in MHB, culture it at 220rpm and 37℃ until the logarithmic growth phase, and adjust its concentration to OD with PBS. 600nm = 0.1, and finally further diluted 1000 times with PBS to 0.5-1×10 5 CFU / mL. (2) Determination of bactericidal kinetics curve: The bacterial solution was mixed with 1×MBC concentration antimicrobial peptide, and 50 μL of samples were diluted at different time points (0, 15s, 30s, 45s, 60s, 3min, 5min, 10min, 15min, 30min), and spread on the corresponding solid culture medium for culture. The bacterial survival rate at each time point was then calculated and the curve was drawn. The test results are shown in Figure

[0050] From the attached Figure 4 It can be seen that at a concentration of 1×MBC, the antimicrobial peptide AG3 killed 100% of Gram-negative bacterial cells within 5 minutes, showing an extremely fast bactericidal rate, indicating that the antimicrobial peptide AG3 has strong bactericidal activity and can be used as an antibiotic alternative in clinical treatment in the future.

[0051] Example 4

[0052] Antibacterial Mechanism of Antimicrobial Peptides

[0053] 1. Cell wall permeability test: This test uses 1-N-phenylnaphthylamine (NPN) uptake test to detect the penetration of peptides into the cell wall of the test strain. The specific steps are as follows: (1) Preparation of bacterial solution: The bacteria in the logarithmic growth phase were collected by centrifugation (5000×g, 5 min), washed three times with 5mM HEPES buffer (pH=7.2, containing 5mM glucose), and resuspended to OD 600nm =0.4 (E. coli), add NPN at a final concentration of 10 μM, and incubate at room temperature in the dark for 30 min. (2) Sample determination: Mix equal volumes of bacterial solution and different concentrations of peptides in a black 96-well plate, and use a fluorescence spectrophotometer to detect the fluorescence intensity of the samples under the conditions of excitation wavelength of 350 nm and emission wavelength of 420 nm.

[0054] The test results are attached. Figure 5 The membrane permeability is greater than 0, which indicates that its antibacterial mechanism is to rupture the outer membrane of the bacteria, thereby exposing the cell contents. At a concentration of 4 μM, the outer membrane permeability reaches more than 50%, which also confirms the high antibacterial activity of the antimicrobial peptide.

[0055] 2. Fluorescence microscopy analysis: E. coli 25922 at the logarithmic growth stage was centrifuged in phosphate buffer and the density was adjusted to 0.5-1.0×10 6 CFU / mL, the dilution treated with 4×MIC AG3 was incubated at 37°C for 1 h, and then propidium iodide (PI) dye (4 μM) was added and incubated for another 30 min. The diluted bacterial solution alone was used as a control. Images were obtained by fluorescence microscopy (EVOS FL Auto, USA). PI dye is an analog of ethidium bromide that releases red fluorescence after intercalation into double-stranded DNA. Although PI cannot pass through the membrane of living cells, it can pass through damaged cell membranes and stain the nucleus.

[0056] See attached for the results Figure 6 The bacterial liquid treated with antimicrobial peptide AG3 showed obvious red light, while no obvious red light was detected in the bacterial liquid used as the control, indicating that antimicrobial peptide AG3 would destroy the cell membrane and cause the bacteria to die.

[0057] The above results show that the designed antimicrobial peptide AG3 not only has excellent antibacterial activity and low hemolytic activity, but also shows strong resistance to various salt ions, indicating that the antimicrobial peptide AG3 has high clinical application potential; at the same time, the membrane-breaking antibacterial mechanism of the antimicrobial peptide AG3 can effectively reduce the possibility of drug resistance of the antimicrobial peptide, and has a high potential to replace antibiotics.

Claims

1. An antimicrobial peptide AG3 with broad-spectrum antimicrobial activity, characterized in that: Its amino acid sequence is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2.

2. The method for preparing the antimicrobial peptide AG3 with broad-spectrum antimicrobial activity according to claim 1, characterized in that: Here are the steps: S1: Design based on the centrosymmetric α-helical peptide template: (y+hhh+y)n-NH2, wherein h is a hydrophobic amino acid, + is a positively charged amino acid, the hydrophobic amino acids are Gly, Ala and Leu, and the positively charged amino acid is Lys, and the amino acid sequence of the obtained polypeptide is shown in SEQ ID No. 1; S2: using a solid phase chemical synthesis method to obtain a peptide resin using a peptide synthesizer, and then cutting the obtained peptide resin with TFA to obtain a polypeptide; S3: After purification by reversed-phase high-performance liquid chromatography and identification by mass spectrometry, the antibacterial activity, antifungal activity, hemolytic activity, salt ion stability, bactericidal kinetics and antibacterial mechanism tests were performed to complete the preparation of the antimicrobial peptide, which was finally named antimicrobial peptide AG3.

3. Use of the antimicrobial peptide AG3 with broad-spectrum antimicrobial activity according to claim 1 in the preparation of a medicament for treating Gram-negative bacteria, Gram-positive bacteria or / and fungal infectious diseases, wherein the Gram-positive bacteria is Staphylococcus aureus; and the fungus is Candida albicans.

4. The use according to claim 3, characterized in that: The Gram-negative bacteria are Escherichia coli, Salmonella typhimurium, Salmonella pullorum or Pseudomonas aeruginosa.

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